荧光定量PCR检测单核细胞增生性李斯特氏菌

目的]建立一种快速检测单核细胞增生性李斯特氏菌的实时荧光定量PCR方法。方法]针对单核细胞增生性李斯特氏菌的特异基因hlyA,结合锁定寡核苷酸（LNA）,设计引物和探针,利用阳性质粒和模拟标本建立单核细胞增生性李斯特氏菌实时定量荧光PCR检测方法。结果]以单核细胞增生性李斯特氏菌为模板克隆了hlyA基因,得到阳性质粒,并进一步建立了荧光定量PCR方法,得到标准曲线Y=-3.273 X＋37.640,相关系数R2=1.000;该方法的灵敏度可以达到1.2×10 CFU/ml;人工污染猪肉检出限可以达到3.2×102 CFU/ml。结论]建立了快速检测单核细胞增生性李斯特氏菌的实时荧光定量PCR方法,为实现食品中单核细胞增生性李斯特氏菌的快速检测构建了一个技术平台。

Detection of Listeria monocytogenes by Real-time Fluorescent PCR

Objective]To develop a rapid and accurate real-time fluorescent PCR method for detection of Listeria monocytogenes.Method]By using hlyA gene of L.monocytogenes as target sequence to design primers and adding four LNA-based TaqMan probe to the primers,a new real-time fluorescent PCR assay was developed and applied to detecting the positive plasmids and the simulated specimens.Result]The hlyA gene was amplified from genomic DNA of Listeria monocytogenes and cloned into pUC119 to generate positive plasmid p...